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D H Dean

Publications and source records attributed to D H Dean.

At least 55 records · Page 3Linked to original sources
PubMed1996

Aminopeptidase N purified from gypsy moth brush border membrane vesicles is a specific receptor for Bacillus thuringiensis CryIAc toxin.

We have evaluated the binding of Bacillus thuringiensis Cry toxins to aminopeptidase N (APN) purified from Lymantria dispar (gypsy moth) brush border membrane vesicle (BBMV). CryIAc toxin bound strongly to APN, while either the structurally related CryIAa and CryIAb toxins or CryIC, CryIIA, and CryIIIA toxins showed weak binding to APN. An in vitro competition binding study demonstrated that the binding of CryIAc to L. dispar BBMV was inhibited by APN. Inhibition of short circuit current for CryIAc, measured by voltage clamping of whole L. dispar midgut, was substantially reduced by addition of phosphatidylinositol-specific phospholipase C, which is known to release APN from the midgut membrane. In contrast, addition of phosphatidylinositol-specific phospholipase C had only a marginal effect on the inhibition of short circuit current for CryIAa. These data suggest that APN is the major functional receptor for CryIAc in L. dispar BBMV. A ligand blotting experiment demonstrated that CryIAc recognized a 120-kDa peptide (APN), while CryIAa and CryIAb recognized a 210-kDa molecule in L. dispar BBMV. In contrast, CryIAa and CryIAb bound to both the 120- and 210-kDa molecules in Manduca sexta BBMV, while CryIAc recognized only the 120-kDa peptide. The 120-kDa peptide (APN) in L. dispar BBMV reacted with soybean agglutinin, indicating that N-acetylgalactosamine is a component of this glycoprotein.

Animals↗
PubMed1995

Domain III exchanges of Bacillus thuringiensis CryIA toxins affect binding to different gypsy moth midgut receptors.

Aminopeptidase-N, purified from gypsy moth (Lymantria dispar L.) brush border membrane vesicles, exhibited specific binding to CryIAc toxin but not to CryIAa toxin. CryIAa-CryIAc hybrid toxins were used to localize the aminopeptidase-N binding region on CryIAc. Slot blot assays and ligand blot experiments demonstrated that the hybrid toxins which have the residues 451 to 623, comprising essentially domain III, from CryIAc toxin exhibited strong binding to purified aminopeptidase-N and 120 kDa brush border membrane protein. In contrast, the hybrid toxins which have the residues 451 to 623 from CryIAa toxin failed to bind to aminopeptidase-N, but did bind to another receptor, a 210 kDa protein. This is the first direct evidence that domain III is involved in receptor binding and the first to demonstrate that domain III substitutions direct the binding of these toxins to different gypsy moth midgut receptors.

Animals↗
PubMed1995

Irreversible binding kinetics of Bacillus thuringiensis CryIA delta-endotoxins to gypsy moth brush border membrane vesicles is directly correlated to toxicity.

To examine the binding of Bacillus thuringiensis delta-endotoxins, CryIAa, CryIAb, and CryIAc, to Lymantria dispar (gypsy moth) brush border membrane vesicles (BBMV), saturation kinetic analyses were conducted according to a two-step interaction scheme [formula: see text] for delta-endotoxin binding to BBMV, rather than the one-step reversible binding presented in prior reports. The order of toxicity of the delta-endotoxins, as measured by the dose required for a 50% inhibition of weight gain (ID50), was CryIAa (77.3 ng) > CryIAb (157 ng) > CryIAc (187 ng). While both the maximum extent of binding, Bmax, and the half-maximum insertion rate concentration, K1/2, was observed to be indirectly related to toxicity, the rate constant of irreversible binding, k2, was found to be directly correlated to toxicity.

Animals↗
PubMed1995

Mutations in domain I of Bacillus thuringiensis delta-endotoxin CryIAb reduce the irreversible binding of toxin to manduca sexta brush border membrane vesicles.

Site-directed mutagenesis was used to generate CryIAb mutants at the selected N-terminal positions to study the function of domain I. Structurally stable mutant proteins were tested for toxicity, receptor binding kinetics, and pore function. Substitutions of tyrosine at position 153 with arginine (Y153R) or alanine (Y153A) did not affect toxicity appreciably, whereas replacing this tyrosine with aspartic acid (Y153D) resulted in a great loss of toxicity. Mutation of alanine at position 92 to glutamic acid (A92E) almost completely abolished toxicity. The initial receptor binding was unchanged as measured by competition binding assays among all mutant proteins. Reduced pore function, however, was observed for mutants A92E and Y153D as tested by voltage clamping. Further studies with specially designed association and dissociation binding assays showed that irreversible binding of these two mutant toxins to Manduca sexta brush border membrane vesicles was significantly reduced. The decrease in irreversible binding was correlated with the changes in toxicity and may reflect a severely disturbed membrane insertion process in these two mutant toxins, leading to reduced pore function and toxicity. The results support the model that domain I is involved in membrane integration and pore formation.

Amino Acid Sequence↗
PubMed1995

Brush border membrane aminopeptidase-N in the midgut of the gypsy moth serves as the receptor for the CryIA(c) delta-endotoxin of Bacillus thuringiensis.

Aminopeptidase-N (AP-N) was purified from gypsy moth (Lymantria dispar, L.) brush border membrane vesicles (BBMV) proteins by mono-Q chromatography and Superdex-75 gel filtration in the presence of the zwitterionic detergent, CHAPS, using FPLC. The purified AP-N, identified by its enzymatic activity, had an apparent size of 100 kDa, and was identified as the unique Bacillus thuringiensis insecticidal toxin, CryIA(c), binding protein. AP-N clearly displayed strong binding to CryIA(c), exhibiting little or no binding to CryIA(a) or CryIA(b), and showing no binding for the coleopteran-specific toxin, CryIIIA. Protein blots of the BBMV proteins probed with biotin-labeled and 125I-labeled insecticidal proteins revealed that CryIAc binds only to 120 kDa protein which is a slightly larger size in comparison to purified AP-N. Antibodies raised against the gypsy moth AP-N demonstrated that the purified AP-N and the 120 kDa CryIA(c) binding protein of total BBMV proteins are antigenically identical.

Amino Acid Sequence↗
PubMed1995

Resistance to Bacillus thuringiensis CryIA delta-endotoxins in a laboratory-selected Heliothis virescens strain is related to receptor alteration.

The Bacillus thuringiensis toxin-binding properties of midgut epithelial cells from two strains of Heliothis virescens were compared. One H. virescens strains (YHD2) which was selected against CryIAc toxin had over 10,000-fold resistance to CryIAc toxin relative to the susceptible strain and was cross-resistant to CryIAa and CryIAb. The second H. virescens strain (YDK) was susceptible to these toxins in the order CryIAc > CryIAb > CryIAa. Receptor-binding properties of CryIAa, CryIAb, and CryIAc toxins were compared between the susceptible and resistant strains. Saturation and competition-binding experiments were performed with brush border membrane vesicles prepared from midguts of the susceptible and resistant insects and 125I-labeled toxins. In the susceptible strain, saturable, specific, and high-affinity binding of all three toxins was observed. The relative binding-site concentration was directly correlated with toxicity (CryIAc > CryIAb > CryIAa). In the resistant strains, the binding affinities of CryIAb and CryIAc were similar to that observed with the susceptible strain and ony minor differences in binding-site concentration (Bmax) were observed. The major difference between the two strains was the total lack of binding of CryIAa toxin to the brush border membrane vesicles of the resistant strain. Heterologous competition-binding experiments and ligand blot analysis supported the hypothesis that there were multiple binding sites for the toxins. On the basis of results of the present study, we propose that alterations in binding proteins shared by all three toxins are a major factor in resistance. This suggests that not all receptors of CryIAc might be involved in toxic function.

Animals↗
PubMed1995

Single amino acid changes in domain II of Bacillus thuringiensis CryIAb delta-endotoxin affect irreversible binding to Manduca sexta midgut membrane vesicles.

Deletion of amino acid residues 370 to 375 (D2) and single alanine substitutions between residues 371 and 375 (FNIGI) of lepidopteran-active Bacillus thuringiensis CryIAb delta-endotoxin were constructed by site-directed mutagenesis techniques. All mutants, except that with the I-to-A change at position 373 (I373A), produced delta-endotoxin as CryIAb and were stable upon activation either by Manduca sexta gut enzymes or by trypsin. Mutants D2, F371A, and G374A lost most of the toxicity (400 times less) for M. sexta larvae, whereas N372A and I375A were only 2 times less toxic than CryIAb. The results of homologous and heterologous competition binding assays to M. sexta midgut brush border membrane vesicles (BBMV) revealed that the binding curves for all mutant toxins were similar to those for the wild-type toxin. However, a significant difference in irreversible binding was observed between the toxic (CryIAb, N372A, and I375A) and less-toxic (D2, F371A, and G374A) proteins. Only 20 to 25% of bound, radiolabeled CryIAb, N372A, and I375A toxins was dissociated from BBMV, whereas about 50 to 55% of the less-toxic mutants, D2, F371A, and G374A, was dissociated from their binding sites by the addition of excess nonlabeled ligand. Voltage clamping experiments provided further evidence that the insecticidal property (inhibition of short-circuit current across the M. sexta midgut) was directly correlated to irreversible interaction of the toxin with the BBMV. We have also shown that CryIAb and mutant toxins recognize 210- and 120-kDa peptides in ligand blotting. Our results imply that mutations in residues 370 to 375 of domain II of CrylAb do not affect overall binding but do affect the irreversible association of the toxin to the midgut columnar epithelial cells of M. sexta.

Amino Acid Sequence↗
PubMed1994

Intracellular proteolysis and limited diversity of the Bacillus thuringiensis CryIA family of the insecticidal crystal proteins.

The current concept of how the highly homologous Bacillus thuringiensis insecticidal crystal protein genes (cry genes) evolved is through recombination among themselves. The cryIA gene family, which is more than 80% identical, consists of only three known genes, even through they are often found together in the same bacterium. To examine the lack of diversity among these genes, recombinatorial chimeric protein toxin genes were constructed and transformed into E. coli, B. subtilis, and B. thuringiensis. Of the nine chimeric proteins examined in this work; three were degraded in E. coli, five in B. subtilis, and seven in B. thuringiensis, suggesting that most Cry proteins resulting from recombination events are degraded by intracellular proteases that are particularly prevalent in B. thuringiensis.

Bacillus subtilis↗
PubMed1994

Location of a lepidopteran specificity region in insecticidal crystal protein CryIIA from Bacillus thuringiensis.

The Bacillus thuringiensis insecticidal crystal protein CryIIA has both high mosquito activity and gypsy moth activity; in contrast CryIIB, which is 87% homologous, displays no mosquito activity and has a three-fold lower gypsy moth activity. The regions responsible for specificity against gypsy moth (Lymantria dispar) and mosquito (Aedes aegypti) larvae were located by introducing MluI and XhoI sites into homologous positions within the putative domain II of both cryIIA and cryIIB genes, which divided almost equally the respective second domains into three regions. Taking advantage of naturally occurring NheI and NarI sites that border the putative domain II, a set of seven chimeric proteins were produced by exchanging all combinations of those regions between CryIIA and CryIIB. Analysis of the toxicity of these chimeric proteins demonstrated that the lepidopteran and dipteran specificity regions of CryIIA were not colinear. While the specificity region of CryIIA against mosquito larvae involved region 1 and probably also region 2, the specificity region of CryIIA against gypsy moth larvae was located within region 2.

Aedes↗
PubMed1994

Identification of amino acid residues of Bacillus thuringiensis delta-endotoxin CryIAa associated with membrane binding and toxicity to Bombyx mori.

Alanine substitution (A3) or deletion (D3) of residues 365 to 371 of Bacillus thuringiensis CryIAa insect toxin removed nearly all toxicity for Bombyx mori (> 1,000-fold less active than the wild type). The loss of larvicidal activity in the mutants was not caused by increased sensitivity to larval gut enzymes but could be attributed to significantly reduced binding to B. mori brush border membrane vesicles. Some or all of the affected amino acid residues may interact directly or indirectly with the B. mori membrane receptor(s). Such receptor binding appears to be directly correlated with insect toxicity.

Amino Acid Sequence↗
PubMed1993

Site-directed mutations in a highly conserved region of Bacillus thuringiensis delta-endotoxin affect inhibition of short circuit current across Bombyx mori midguts.

Bacillus thuringiensis delta-endotoxins (Cry toxins) are insecticidal proteins of approximately 65 kDa in the proteolytically processed and active form. The structure of one of these toxins, CryIIIA, has been determined by Li et al. [Li, J., Carroll, J. & Ellar, D. J. (1991) Nature (London) 353, 815-821] and contains three domains. It is believed that other delta-endotoxins adopt similar three-dimensional structure. Li et al. proposed that the first domain is the membrane pore-forming domain. Previous work from our laboratory has shown that the second domain is the receptor binding domain, but the function of the third domain is unclear. Site-directed mutagenesis was used to convert the "arginine face" of one of five highly conserved regions, QRYRVRIRYAS of CryIAa (residues 525-535), to selected other residues. This sequence corresponds to the beta-sheet 17 of CryIIIA in the third domain. Mutations in the second and third arginine positions resulted in structural alterations in the protein and were poorly expressed in Escherichia coli. Toxins from genes mutated to replace lysine for the first and fourth arginines were unaltered in expression and structure, as measured by trypsin activation, CD spectra, and receptor binding, but were substantially reduced in their insecticidal properties and inhibition of short circuit current across Bombyx mori midguts. It is proposed that this region plays a role in toxin function as an ion channel.

Amino Acid Sequence↗
PubMed1993

Suppression of protein structure destabilizing mutations in Bacillus thuringiensis delta-endotoxins by second site mutations.

Reciprocal exchange of a small region (residues 428-450) within the specificity determining region of two Bacillus thuringiensis delta-endotoxins, CryIAa and CryIAc, resulted in two recombinant proteins that possess a decreased insecticidal activity to Bombyx mori and Manduca sexta. Site-directed mutations introduced in this region of one of the recombinant proteins, with the intent of restoring insecticidal activity, resulted in further reduction of toxicity. We determined that the loss of insecticidal activity in the mutants and the original recombinants was associated with altered toxin protein structure, as measured by sensitivity to intracellular and exogenous proteases. The structural instability of the site-directed mutant proteins could be suppressed genetically by subcloning the mutated region into cryIAc or by introducing second site mutations in defined regions of the mutated cryIAa gene. The second site mutations, by themselves, also produced unstable proteins. In the course of this study, we demonstrated that this small region does not suffice as a specificity determining region for M. sexta.

Animals↗
PubMed1993

Structural stability of Bacillus thuringiensis delta-endotoxin homolog-scanning mutants determined by susceptibility to proteases.

Forty homolog-scanning (double-reciprocal-crossover) mutant proteins of two Bacillus thuringiensis delta-endotoxin genes (cryIAa and cryIAc) were examined for potential structural alterations by a series of proteolytic assays. Three groups of mutants could be identified. Group 1, consisting of 13 mutants, showed no delta-endotoxin present during overexpression conditions in Escherichia coli (48 h at 37 degrees C, with a ptac promoter). These mutants produced full-sized delta-endotoxin detectable by polyacrylamide gel electrophoresis with Coomassie blue staining or Western immunoanalysis after 24 h of growth but not after 48 h, suggesting sensitivity to intracellular proteases. Group 2 consisted of 13 mutants that produced stable delta-endotoxins that were completely digested by 2% bovine trypsin. In contrast, native delta-endotoxin produces a 65,000-Da trypsin-resistant peptide, which is the active toxin. Group 3 mutants expressed delta-endotoxin and trypsin-stable toxins, similar to the wild type. In this study, 12 group 3 mutant toxins were compared with wild type toxins by thermolysin digestion at a range of temperatures. The two wild-type toxins exhibited significant differences in thermolysin digestion midpoints. Among the group 3 mutants, most possessed significantly different protein stabilities relative to their parental toxins. Two of the group 3 mutants were observed to have exchanged the thermolysin sensitivity properties of the parental toxins.

Bacillus thuringiensis↗
PubMed1993

Managing the care of patients with mental retardation: a survey of physicians.

A questionnaire regarding the clinical management of patients with mental retardation living in the community was mailed to a 50% random sample of physicians practicing in Maine; the response rate was 56%. Almost 80% of respondents reported that they were currently providing care to patients with mental retardation. Survey results identified the major issues confronting physicians treating such patients and quantitatively assessed their importance from the physicians' perspective. Inadequate medical information was identified as the greatest obstacle to patient care. Physicians' ratings of the utility of specific measures designed to lessen these obstacles were presented. Generally, continuing education was viewed as less useful than practice support measures.

Activities of Daily Living↗
PubMed1992

Expression of a gene encoding a scorpion insectotoxin peptide in yeast, bacteria and plants.

The nucleotide sequence encoding the scorpion insectotoxin I5A was chemically synthesized and expressed in yeast, bacteria and tobacco. The I5A peptides produced in these organisms were purified using an immunoaffinity chromatography procedure. I5A produced using the bacterial secretion system was efficiently secreted and released into the culture medium. In contrast, only a trace amount of I5A was detected in bacterial cytosols when expressed from a direct expression vector, suggesting that I5A was unstable in bacterial cells. I5A secreted from yeast using an alpha-factor signal sequence was shown to have an N-terminal (Glu-Ala)2 extension, indicating incomplete processing of the secreted peptide by dipeptidyl aminopeptidase A. In tobacco, a nonsecreted form of the protein was produced. No measurable insect toxicity was observed when insect larvae were assayed, regardless of whether I5A was produced in yeast, bacteria or tobacco. The lack of toxicity is almost certainly the result of improper folding due to incorrect disulfide bond formation. The inability to produce a biologically active peptide must be overcome before scorpion toxins might be used for the genetic engineering of plants for insect resistance. The yeast and bacterial expression systems described here may be useful for further studies on the problem of expressing a biologically active peptide.

Amino Acid Sequence↗
PubMed1992

Location of a Bombyx mori receptor binding region on a Bacillus thuringiensis delta-endotoxin.

Receptor binding studies were performed with 125I-labeled trypsin-activated insecticidal toxins, CryIA(a) and CryIA(c), from Bacillus thuringiensis on brush-border membrane vesicles (BBMV) prepared from Bombyx mori larval midgut. Bioassays were performed by gently force feeding B. mori with diluted toxins. CryIA(a) toxin (LD50; 0.002 micrograms) was 200 times more active against B. mori larvae than CryIA(c) toxin (LD50; 0.421 micrograms) and showed high-affinity saturable binding. The Kd and the binding site concentration for CryIA(a) toxin were 3.5 nM and 7.95 pmol/mg, respectively. CryIA(c) toxin (Kd, 50.35 nM; Bmax, 2.85 pmol/mg) did not demonstrate high-affinity binding to B. mori BBMV. Control experiments with CryIA(a) and CryIA(c) toxins revealed no binding to mouse small intestine BBMV and nonspecific binding to pig kidney BBMV. These data provide evidence that binding to a specific receptor on the membrane of midgut epithelial cells is an important determinant with respect to differences in insecticidal spectrum of insecticidal crystal proteins. To locate a B. mori receptor binding region on the CryIA(a) toxin, homologous and heterologous competition binding studies were performed with a set of mutant proteins which had previously been used to define the B. mori "specificity domain" on this toxin (Ge, A. Z., Shivarova, N. I., and Dean, D. H. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 4037-4041). These mutant proteins have had regions of their genes reciprocally exchanged with the cryIA(c) gene. A B. mori receptor binding region on CryIA(a) toxin includes the amino-terminal portion of the hypervariable region, amino acids 332-450, which is identical to the previously described B. mori specificity determining region. These data provide direct evidence that delta-endotoxins contain a tract of amino acids that comprise a binding region and as a results determines the specificity of a toxin.

Amino Acid Sequence↗
PubMed1991

Functional domains of Bacillus thuringiensis insecticidal crystal proteins. Refinement of Heliothis virescens and Trichoplusia ni specificity domains on CryIA(c).

Insecticidal crystal proteins (delta-endotoxins), CryIA(a) and CryIA(c), from Bacillus thuringiensis are 82% homologous. Despite this homology, CryIA(c) was determined to have 10-fold more insecticidal activity toward Heliothis virescens and Trichoplusia ni than CryIA(a). Reciprocal recombinations between these two genes were performed by the homolog-scanning technique. The resultant mutants had different segments of their primary sequences exchanged. Bioassays with toxin proteins from these mutants revealed that amino acids 335-450 on CryIA(c) are associated with the activity against T. ni, whereas amino acids 335-615 on the same toxin are required to exchange full H. virescens specificity. One chimeric protein toxin, involving residues 450-612 from CryIA(c), demonstrated 30 times more activity against H. virescens than the native parental toxin, indicating that this region plays an important role in H. virescens specificity. The structural integrity of mutant toxin proteins was assessed by treatment with bovine trypsin. All actively toxic proteins formed a 65-kDA trypsin-resistant active toxic core, similar to the parental CryIA(c) toxin, indicating that toxin protein structure was not altered significantly. Contrarily, certain inactive mutant proteins were susceptible to complete protease hydrolysis, indicating that their lack of toxicity may have been due to structural alterations.

Bacillus thuringiensis↗